pET vector cloning of hydrophobic protein gene

hong dang hdang at channel.neusc.bcm.tmc.edu
Wed Aug 2 14:26:28 EST 1995


> In article <3vltmi$qn5 at warp.cris.com>, mike.holloway at stjude.org (Mike
> Holloway) wrote:
> 
> > I'm giving up on the pET system and wondered if anyone else has had 
> > similar experiences.  Even with the thioredoxin fusion protein vector, 
> > I can not isolate a transformant with my sequence and an intact vector. 
> > I do get many rearrangements - things that are not whole pET vector or 
> > PCR template from the preparation of my insert.  The transformants are of
> > various sizes, with various restriction sites missing.  Other cloning 
> > projects using the same bugs and reagents are working just fine, without
> > any evidence of contamination. 
> > 
> > I have to conclude that some protein is being made, despite what anyone 
> > says about the T7 promoter not being used without the polymerase being 
> > pumped out, and that its toxic.  This has been the previous experience 
> > in our lab with this protein using other bacterial expression vectors.  
> > Apparently this isn't an isolated experience, since several companies 
> > have recently come out with this thioredoxin fusion protein scheme that's 
> > supposed to solve everything.  
> > 
> > Anyone had experience with pET vectors?
> > 
> > =========================================================================
> >=============

There may be 1 way out: recently people in our lab has succeeded in
cloning one of
those "toxic" thing in a pET, which had failed before.
Here's the trick: a PCR product was direction-cloned into both pET-20b and
pET25b,
1st one, no colony, 2nd one, many. The relevant difference, there is a
Lac-op following
T7 promoter in 25b. So in addition to controlling T7 pol, 25b also has a
lac-repression
on your gene of interest that may further cut down on leaky expression of
the thing
and make it tolerable for the bug. 
So seems there is a dose effect, if your protein is really, really toxic
to the bug, it 
still may not express. But try the T7-lac combo vector before giving it
up, since you
might hit the same problem with another system as well.

Hong

-- 
Hong Dang (hdang at channel.neusc.bcm.tmc.edu)

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